The human leukocyte antigen (HLA) plays a central role in immunological discrimination between self and non-self. This discrimination is achieved when T cells recognize, via T cell receptors, HLA-peptide complexes presenting self- or non-self-derived peptides on HLAs. T cells recognize cells expressing, on the surface, HLA-peptide complexes presenting non-self (pathogenic microbes such as viruses and bacteria, or foreign antigens such as pollens)-derived peptides on self-HLAs, or cells expressing, on the surface, non-self HLA alleles that have entered the body through transplantation or transfusion, thereby causing activation of immunocytes or destroy of the presenting cells.
Such activation of immunocytes or destroy of the presenting cells causes a rejection response or graft versus host disease (GVHD) in transfusion, medical transplantation including bone marrow transplantation, or regenerative medicine using iPS cells or ES cells. In patients receiving frequent platelet transfusion, an antibody against a non-self HLA is produced and brings about a significant reduction in the efficacy of the platelet transfusion. In some cases of medication, a drug (and a peptide) bound with a particular HLA may be recognized as a foreign substance, causing a severe adverse drug reaction based mostly on an allergy response.
Accordingly, medical transplantation or regenerative medicine requires matching of HLAs between a patient and a donor. Transfusion of “HLA-compatible platelet” with HLA match is also necessary for platelet transfusion patients in which an anti-HLA antibody against a particular allele is produced. For adverse drug reactions, it is also important to examine HLAs before medication when the drug to be administered is reportedly related to a particular HLA allele. In actuality, package inserts of some drugs clearly states recommendation to examine HLAs. Peptide vaccine therapy of cancer also requires examining HLAs for predicting whether or not the peptide vaccine can bind to patient's HLAs.
As major HLAs, six types of antigens are known, namely, class I molecules (HLA-A, HLA-B and HLA-C), which are expressed in almost all cells, and class II molecules (HLA-DR, HLA-DQ and HLA-DP), which are expressed mainly in immune cells.
The HLA class I antigen consists of a highly polymorphic α chain and a substantially non-polymorphic β2-microglobulin; whereas the HLA class II antigen consists of a highly polymorphic β chain and a less polymorphic α chain. The α chains of class I molecules are encoded by HLA-A, HLA-B and HLA-C genes. The β chains of class II antigens are encoded by HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 and HLA-DPB1 genes, whereas the α chains are encoded by HLA-DRA1, HLA-DQA1 and HLA-DPA1 genes. In a gene level, in HLA class I antigens, exon 2 and exon 3 of a gene encoding an α chain are highly polymorphic; whereas, in HLA class II antigens, exon 2 of a gene encoding a β chain is highly polymorphic.
A gene region encoding an HLA is located on short arm of human chromosome 6 at 6p21.3. A class I region (HLA-A, HLA-C, HLA-B, etc.), a class III region and a class II region (HLA-DRA, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, etc.) are arranged in this order from the telomere side toward the centromere side. Many genes are encoded at an extremely high density and association of these genes with transfusion, transplantation and various diseases has been reported. In the class III region, no HLA genes are present and genes of complement components and tumor necrosis factors (TNF), etc. are present.
In an HLA-DRB gene region encoding a β chain of an HLA-DR antigen, it has been confirmed that 5 types of structural polymorphisms are present. In DR1 type and DR10 type, pseudogenes such as HLA-DRB6 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome. In DR2 type, an HLA-DRB5 (DR51) gene and pseudogenes such as HLA-DRB6 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome. In DR3, DR5 and DR6 types, an HLA-DRB3 (DR52) gene and pseudogenes such as HLA-DRB2 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome. In DR4, DR7 and DR9 types, an HLA-DRB4 (DR53) gene and pseudogenes such as HLA-DRB7, HLA-DRB8 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome. In contrast to these, in DR8 type, no HLA-DRB genes except HLA-DRB1 are located on the same chromosome (see FIG. 1).
In the exon of each allele, a plurality of regions exhibiting polymorphism are present. In many cases, a nucleotide sequence (amino acid sequence) present in a certain polymorphic region is commonly present in a plurality of alleles. In short, each HLA allele is specified by a plurality of polymorphic regions in combination. In an HLA class II antigen, not only a polymorphic region in the exon but also exon 2 or exon 3 having the same nucleotide sequence is sometimes commonly present in a plurality of alleles.
Since a highly polymorphic region is present in an HLA, the number of types of alleles is known to be extremely large and notation of them has been defined: i.e., a first field (two-digit level) is for discrimination of serologic HLA types, a second field (4-digit level) is for discrimination of alleles having an amino acid substitution in the same serologic HLA type, a third field (6-digit level) is for discrimination of alleles having a base substitution not accompanying an amino acid mutation and a fourth field (8-digit level) is for discrimination of alleles having a base substitution in an intron, which is out of the genetic region encoding an HLA molecule (see FIG. 2).
In various medical cases, examination of HLA alleles (DNA typing of HLA genes) is important. However, a SBT (sequence-based typing) method or a Luminex method (PCR-sequence-specific oligonucleotide probes (SSOP)-Luminex method), which has heretofore been frequently used, cannot easily determine whether polymorphic regions are in a cis-configuration (on the same chromosome) or in a trans-configuration (on different chromosomes), if a plurality of different polymorphic regions are present between alleles. Therefore, the alleles were sometimes unable to be accurately determined due to the occurrence of so-called phase ambiguity.
Hence, we developed a method capable of eliminating phase ambiguity by using a next generation sequencer (high throughput massive parallel sequencer). In this method, however, typing of fragmented DNAs in a poor state of preservation was sometimes not easily performed because a long region containing 3′UTR was amplified from a promoter region. Particularly, an HLA class II gene has long intron 1, which requires amplifying a region about twice longer than that of a class I gene. Therefore, typing of DNA fragment samples was not easily performed.